Recombinant Coxiella burnetii Elongation factor Ts (tsf)

What is Elongation factor Ts in Coxiella burnetii and what is its function?

Elongation factor Ts (EF-Ts) in Coxiella burnetii is an essential component of the bacterial translational machinery. It functions as a guanine nucleotide exchange factor that interacts with Elongation factor Tu (EF-Tu) to facilitate protein synthesis. EF-Ts is encoded by the tsf gene and has been identified as differentially expressed during the bacterium's biphasic developmental cycle, with upregulated expression in the Large Cell Variant (LCV) form compared to the Small Cell Variant (SCV) form . This differential expression pattern suggests EF-Ts plays a crucial role in the metabolically active phase of C. burnetii's life cycle.

What experimental methods have been used to identify and characterize C. burnetii EF-Ts?

Multiple complementary approaches have been employed to characterize C. burnetii EF-Ts:

• Monoclonal antibody screening: MAb NM7.3 was used to identify a ~32-kDa LCV-upregulated antigen, which was subsequently identified as EF-Ts through λZapII C. burnetii DNA expression library screening .

• Western blotting: Used to compare reactivity with LCV and SCV antigens, demonstrating the differential expression of EF-Ts between morphological forms .

• Densitometric analysis: Employed to quantify the relative levels of EF-Ts expression by separating equal amounts of organisms (measured by optical density at 600 nm) .

• Mass spectrometry: Whole-cell mass spectrometry has been used to identify and quantify proteins in different developmental forms of C. burnetii .

How is EF-Ts expression regulated during the C. burnetii developmental cycle?

The expression of EF-Ts is upregulated in the Large Cell Variant (LCV) compared to the Small Cell Variant (SCV) of C. burnetii. This differential expression pattern correlates with the metabolic state of the bacterium during its biphasic developmental cycle . The LCV represents the metabolically active, replicating form, while the SCV is the non-replicating, resistant form that enables environmental persistence.

Research suggests that the alternative sigma factor RpoS may play a role in regulating this differential expression. RpoS functions as a global regulator in C. burnetii, affecting a substantial portion of the genome (>25%) during developmental transitions, particularly during SCV development . While direct regulation of tsf by RpoS has not been explicitly demonstrated, the pattern of differential expression is consistent with the broader regulatory network controlled by RpoS during C. burnetii development.

What is the relationship between RpoS and tsf expression in C. burnetii?

While the search results do not directly establish a regulatory relationship between RpoS and tsf, several lines of evidence suggest potential connections:

• RpoS globally regulates genes within the C. burnetii genome, with a major subset associated with SCV production during the biphasic development cycle .

• Studies with a C. burnetii ΔrpoS mutant revealed significant transcriptional changes affecting stress responses, amino acid acquisition, cell wall remodeling, and type 4B secretion system assembly .

• The ΔrpoS mutant showed defective intracellular replication when infecting cells in the SCV form but not in the LCV form, suggesting RpoS-regulated genes are crucial for developmental transitions .

Given that EF-Ts shows differential expression between LCV and SCV forms, it is reasonable to hypothesize that RpoS may influence tsf expression directly or indirectly through its extensive regulatory network.

What methodological approaches can researchers use to study the differential expression of tsf?

Researchers investigating the differential expression of tsf in C. burnetii can employ several complementary approaches:

• Transcriptional analysis:

  • RNA sequencing to compare tsf mRNA levels between wild-type and mutant strains (e.g., ΔrpoS)

  • Quantitative RT-PCR to validate expression differences

  • Promoter-reporter fusions to identify regulatory elements

• Protein-level analysis:

  • Western blotting with specific antibodies (e.g., MAb NM7.3)

  • Quantitative proteomics using mass spectrometry

  • Immunofluorescence microscopy to visualize expression patterns in situ

• Functional studies:

  • Creation of tsf deletion or conditional mutants

  • Complementation studies to confirm phenotypes

  • Protein-protein interaction studies (pull-downs, co-immunoprecipitation)

What are the optimal conditions for expressing and purifying recombinant C. burnetii EF-Ts?

Based on available data, researchers should consider the following for optimal expression and purification of recombinant C. burnetii EF-Ts:

• Expression system: Yeast expression systems have been successfully used for C. burnetii EF-Ts production . The commercially available recombinant protein (CSB-YP025124DXO) is produced in yeast with >85% purity as determined by SDS-PAGE .

• Construct design: Full-length protein (amino acids 1-296) with appropriate tags for purification has been successfully expressed .

• Purification strategy:

  • Affinity chromatography using appropriate tags

  • Additional purification steps to achieve high purity (>85% for most applications)

  • Consider tag removal if it might interfere with functional studies

• Storage conditions:

  • Store at -20°C or -80°C for extended storage

  • Avoid repeated freezing and thawing

  • Store working aliquots at 4°C for up to one week

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol for long-term storage

How can researchers assess the functional activity of recombinant EF-Ts?

Researchers can evaluate the functional activity of recombinant C. burnetii EF-Ts through several experimental approaches:

• GDP/GTP exchange assay:

  • Measure the ability of EF-Ts to promote nucleotide exchange on EF-Tu

  • This can be quantified using fluorescently labeled nucleotides or radioactive assays

• Protein-protein interaction studies:

  • Surface plasmon resonance to measure binding kinetics with EF-Tu

  • Isothermal titration calorimetry for thermodynamic parameters

  • Pull-down assays to confirm interaction with EF-Tu and potentially identify other binding partners

• Structural validation:

  • Circular dichroism to confirm proper folding

  • Limited proteolysis to assess conformational integrity

  • Thermal shift assays to evaluate stability under different conditions

• Cell-based complementation:

  • Test ability to restore function in heterologous systems with conditional tsf mutants

What methodological considerations are important when using recombinant EF-Ts for structural studies?

When using recombinant C. burnetii EF-Ts for structural studies, researchers should consider:

• Protein quality requirements:

  • Higher purity (>95%) than typically needed for biochemical assays

  • Monodispersity assessment by dynamic light scattering

  • Batch consistency verification by mass spectrometry

• Buffer optimization:

  • Screen multiple buffer conditions for optimal stability

  • Assess aggregation propensity at concentrations needed for structural work

  • Consider additives that may enhance stability without interfering with structure

• Complex formation considerations:

  • For co-crystallization studies with partners like EF-Tu, optimize complex formation conditions

  • Characterize complex stability and stoichiometry

• Technique selection based on specific research questions:

  • X-ray crystallography for high-resolution static structures

  • NMR for dynamics and solution behavior

  • Cryo-EM for larger complexes or challenging crystallization targets

How can researchers use recombinant EF-Ts to investigate C. burnetii pathogenesis?

Recombinant C. burnetii EF-Ts offers several applications for studying pathogenesis:

• Developmental cycle studies:

  • As a marker for LCV-SCV transitions due to its differential expression

  • In combination with RpoS studies to understand regulatory networks controlling development

• Host-pathogen interaction analysis:

  • Investigate potential interactions between EF-Ts and host factors

  • Examine the impact of EF-Ts antibodies on intracellular replication

• Comparative studies with other intracellular pathogens:

  • Compare the properties and regulation of EF-Ts with homologs in related bacteria like Legionella pneumophila

  • Identify unique features that might contribute to C. burnetii's distinctive intracellular lifestyle

• Integration with global regulatory analyses:

  • Use recombinant EF-Ts in chromatin immunoprecipitation experiments to identify potential regulatory proteins

  • Combine with transcriptomic and proteomic approaches to place EF-Ts in the context of broader regulatory networks

What experimental strategies can address contradictory findings about EF-Ts function?

When faced with contradictory findings regarding EF-Ts function, researchers should consider:

• Developmental stage-specific analyses:

  • Separately examine EF-Ts function in LCV versus SCV forms

  • Use synchronized cultures to distinguish stage-specific effects

  • Create conditional mutants that allow temporal control of EF-Ts expression

• Host cell type considerations:

  • Compare EF-Ts expression and function in different host cell types (e.g., Vero cells versus THP-1 macrophages)

  • Assess whether host factors influence EF-Ts activity

• Strain variation analysis:

  • Compare EF-Ts sequences and expression patterns across different C. burnetii strains

  • Determine whether observed functional differences correlate with genetic variation

• Methodological reconciliation:

  • Directly compare different experimental approaches side-by-side

  • Standardize growth conditions, bacterial preparation methods, and analytical techniques

  • Develop consensus protocols for C. burnetii research

How might EF-Ts interact with the stress response system in C. burnetii?

The relationship between EF-Ts and stress response systems in C. burnetii presents an intriguing research direction:

• Connection to RpoS regulation:

  • RpoS regulates genes involved in stress responses, and its mutation affects resistance to oxidative stress and cell wall integrity

  • The differential expression of EF-Ts between LCV and SCV forms may reflect adaptation to different stress environments

• Experimental approaches to investigate this interaction:

  • Examine EF-Ts expression under various stress conditions (oxidative stress, nutrient limitation, pH changes)

  • Compare stress resistance phenotypes between wild-type and tsf mutant strains

  • Investigate whether EF-Ts has moonlighting functions beyond its canonical role in translation

• Integration with broader cell physiology:

  • Analyze how translational regulation through EF-Ts connects to stress response pathways

  • Determine whether EF-Ts participates in selective translation of stress-response proteins

• Comparative analysis with related bacteria:

  • Investigate whether the connection between EF-Ts and stress responses is conserved in related intracellular pathogens

  • Identify unique aspects of C. burnetii's use of translational regulation for stress adaptation

What are the most promising approaches for using EF-Ts as a potential therapeutic target?

While direct therapeutic applications are not discussed in the search results, several research approaches could explore EF-Ts as a potential intervention target:

• Structure-based drug design:

  • Utilize the known sequence and predicted structure of C. burnetii EF-Ts to identify unique features for selective targeting

  • Focus on the EF-Ts/EF-Tu interaction interface as a potential site for disruption

• Functional inhibition strategies:

  • Screen for small molecules that inhibit the nucleotide exchange activity of EF-Ts

  • Develop peptide mimetics that compete with natural binding partners

• Vaccine development considerations:

  • Assess the immunogenicity of recombinant EF-Ts

  • Determine whether antibodies against EF-Ts can neutralize C. burnetii infection

  • Evaluate EF-Ts as a potential component of subunit vaccines

• Delivery system development:

  • Design approaches to deliver inhibitors to the intracellular niche where C. burnetii resides

  • Explore host-directed therapies that might indirectly affect EF-Ts function

How can researchers design experiments to clarify the role of EF-Ts in developmental transitions?

To better understand EF-Ts's role in C. burnetii developmental transitions, researchers could:

• Generate conditional or inducible tsf mutants:

  • Create strains with controlled expression of EF-Ts to observe effects on developmental transitions

  • Compare phenotypes with those of the ΔrpoS mutant, which shows defects in SCV-mediated infection

• Temporal expression analysis:

  • Perform time-course studies tracking EF-Ts levels during the LCV-to-SCV transition

  • Correlate changes in EF-Ts expression with morphological and physiological changes

• Localization studies:

  • Use fluorescently tagged EF-Ts to track its subcellular localization during developmental transitions

  • Determine whether EF-Ts shows differential localization patterns in LCV versus SCV forms

• Interactome analysis:

  • Identify EF-Ts protein interaction partners in both LCV and SCV forms

  • Determine whether these interactions change during developmental transitions

• Combined multi-omics approach:

  • Integrate transcriptomic, proteomic, and metabolomic data to place EF-Ts in the context of global changes during development

  • Identify potential regulatory networks controlling EF-Ts expression and function

What experimental designs could assess potential non-canonical functions of EF-Ts in C. burnetii?

Beyond its canonical role in translation, EF-Ts might have additional functions in C. burnetii that could be investigated through:

• Protein-protein interaction screening:

  • Perform systematic screens (yeast two-hybrid, pull-downs, cross-linking coupled with mass spectrometry) to identify unexpected interaction partners

  • Focus particularly on partners unique to C. burnetii or intracellular pathogens

• Subcellular localization studies:

  • Determine whether EF-Ts localizes to unexpected cellular compartments under specific conditions

  • Compare localization patterns between LCV and SCV forms

• Post-translational modification analysis:

  • Identify potential modifications of EF-Ts that might regulate non-canonical functions

  • Determine whether modifications differ between developmental stages

• Heterologous expression studies:

  • Express C. burnetii EF-Ts in model organisms and assess phenotypic effects

  • Compare with effects of EF-Ts from other bacterial species

• Domain mapping experiments:

  • Create truncation or point mutation variants to identify regions required for potential non-canonical functions

  • Distinguish domains required for translation versus other cellular processes